Labelling of Oligonucleotides
Fluorescent, non-radioactive labelling is a highly desirable method for the detection of nucleic acids. For example, this method is useful in automated DNA sequencing, in situ detection of hybridization, detection of PCR products, structural studies, and any of several other applications. In the past, labelling of oligonucleotides has been most conveniently accomplished on an automated synthesizer by introduction of a derivatized deoxyuridine amidite.sup.1 or a linker amidite bearing a protected primary amine.sup.2,3 or thiol..sup.4 (By "oligonucleotide" we mean an oligomer of DNA, RNA, or modifications thereof, in the range of 3 to 200 bases in length.) Consequently, preparation of a labelled oligonucleotide requires synthesis of an oligonucleotide bearing the aforementioned modifications. The oligomer is deprotected, liberating the nucleophile, which can react with a fluorescent label. This procedure entails at least a partial purification of the deprotected oligomer, reaction with the fluorescent dye derivative, removal of the excess reagent, and purification of the labelled oligomer. The purification of the product is often tedious and addition of the label increases synthesis time considerably. The overall process requires approximately two days, compared to less than one day for preparation of the unlabelled oligonucleotide.
Current procedures for the preparation of other types of non-radioactively labelled oligonucleotides usually require similar procedures..sup.5 In general, after deprotection and purification, the liberated nucleophile is coupled with a label (fluorescent, bioreactive, chemiluminescent, photolabile, etc.).
A few examples of labelling amidites, which enable direct attachment to the oligonucleotide, have been reported. An amidite bearing a chemically inert bathophenanthroline-ruthenium complex has been prepared and proposed as a fluorescent label for DNA sequencing and as a diagnostic probe..sup.6 Biotin-containing amidites have been prepared by several groups,.sup.7,8 and Teoule and co-workers have prepared amidites with dinitrophenyl, dansyl, and pyrenyl labels..sup.8 Du Pont workers have also reported the synthesis of succinylxanthene-labelled amidites..sup.9 Cech and co-workers have published the synthesis of a fluorescein amidite where the linker side-arm is attached to one of the phenolic hydroxyls..sup.10
Indocarbocyanine Dyes
Indocarbocyanines have long been known for their dye properties..sup.11 Recently some indocarbocyanine derivatives have proved to be extremely fluorescent and are quite useful in the labelling of biomolecules..sup.12,13a-d Because of the low background fluorescence of biological materials in the longer wavelength portion of the spectrum, the signal-to-noise ratio of analyses using these dyes is very good.
It is usually advantageous to use as little of labelled probe as possible in order not to perturb the biological system or process with the probe. Therefore, the better the signal-to-noise ratio, the less probe is necessary. Indocarbocyanines have a very high absorbance and are among the "brightest" of the common dyes used in biological labelling..sup.14
Indocarbocyanines have been introduced into proteins via carboxamide.sup.13b,14 and thiourea.sup.15 linkages. One method.sup.12 of linking an indocarbocyanine to a nucleic acid used the introduction of an amino linker to the 5'-end of the oligonucleotide. An N-hydroxysuccinimide (NHS) ester of the indocarbocyanine carboxylic acid was added to an amino-linked oligonucleotide and the resulting conjugate isolated and purified. An alternative procedure would involve the addition of a linker phosphoramidite to the 5' end of an oligonucleotide on a DNA synthesizer. After deprotection and desalting of the phosphoramidite-linked oligonucleotide, the active nucleophile then reacts with the indocarbocyanine-carboxylic acid NHS ester to yield labelled oligonucleotide. The groups present in the linker are preferred because they are stable to conditions used in DNA synthesis, sequencing, and hybridization studies, as well as stability in long-term storage.
In spite of the long time for reaction and workup (compared to the synthesis of the oligonucleotide), generally introduction of labels has been done after deprotection of the oligonucleotide. Reaction and workup normally requires two days.
For these reasons, there is a need in the art of oligonucleotide labelling for an indocarbocyanine-linked phosphoramidite which allows the labelling of oligonucleotides in one step.